Divinylpyrimidine reagents generate antibody–drug conjugates with excellent in vivo efficacy and tolerability

The development of divinylpyrimidine (DVP) reagents for the synthesis of antibody–drug conjugates (ADCs) with in vivo efficacy and tolerability is reported. Detailed structural characterisation of the synthesised ADCs was first conducted followed by in vitro and in vivo evaluation of the ADCs’ ability to safely and selectively eradicate target-positive tumours.


ADC Synthesis
To a solution of trastuzumab (900 μL, 20.41 μM, 3.0 mg/mL) in TBS (25 mM Tris HCl pH 8, 25 mM NaCl, 0.5 mM EDTA) was added TCEP (10 equiv.). The mixture was vortexed and incubated at 37 °C for 1 h. A solution of DVP 1 (20 mM in DMSO, adjusted to 10% v/v) was added (40 equiv.) and the reaction mixture incubated at 37 °C for 2 h. The excess reagents The reaction product was completely buffer exchanged into PBS and concentrated by repeated diafiltration using an Amicon-Ultra centrifugal filter (10,000 MWCO, Merck Millipore). were removed by two successive PD-10 columns (GE Healthcare) pre-equilibrated with PBS.
The reaction product was completely buffer exchanged into PBS and concentrated by repeated diafiltration using an Amicon-Ultra centrifugal filter (10,000 MWCO, Merck Millipore).

Hydrophobic Interaction Chromatography (HIC)
Hydrophobic interaction chromatography (HIC) was carried out on an Agilent 1200 Series system using a Tosoh Bioscience TSKgel Butyl-NPR (3.5 cm ´ 4.6 mm, 2.5 µm particle size) column at a flow rate of 0.6 mL/min and a gradient of 0-100% B over 20 minutes (Solvent A: 1.5M ammonium sulfate, 25 mM NaPi, pH 7; Solvent B: 25% isopropyl alcohol in 25 mM NaPi, pH 7). 10 µg of trastuzumab or ADC (1-2 mg/mL in PBS) was analysed per run. Average DAR for each ADC was calculated as follows, where DARn corresponds to the peak area at 280 nm

Size Exclusion Chromatography (SEC)
Size-exclusion chromatography (SEC) was carried out on an Agilent 1200 Series system using a TSKgel G3000SWXL (30 cm ´ 7.8 mm, 5 µm particle size) with a mobile phase of PBS (50 mM sodium phosphates, 100 mM NaCl, 0.02% sodium azide, pH 7.0) at a flow rate of 0.5 mL/min over 30 min. 10 µg of trastuzumab or ADC (1-2 mg/mL in PBS) was analysed per run. Samples were analysed via absorption at 280 nm.

Liquid Chromatography-Mass Spectrometry (LCMS)
Protein LCMS was performed on a Xevo G2-S TOF mass spectrometer coupled to an Acquity UPLC system using an Acquity UPLC BEH300 C4 column (1.7 μm, 2.1 × 50 mm). H2O with 0.1% formic acid (solvent A) and 95% MeCN and 5% water with 0.1% formic acid (solvent B), were used as the mobile phase at a flow rate of 0.2 mL/min. The gradient was programmed as follows: 95% A for 0.93 min, then a gradient to 100% B over 4.28 min, then 100% B for 1.04 minutes, then a gradient to 95% A over 1.04 min. The electrospray source was operated with a capillary voltage of 2.0 kV and a cone voltage of 150 V. Nitrogen was used as the desolvation gas at a total flow of 850 L/h. Total mass spectra were reconstructed from the ion series using the MaxEnt algorithm preinstalled on MassLynx software (v4.1 from Waters) according to the manufacturer's instructions. Trastuzumab samples were deglycosylated with PNGase F (New England Biolabs) prior to LCMS analysis. supplemented with 10% FBS, 2 mM L-glutamine, 50 U/mL penicillin and 50 μg/mL streptomycin. BT474 cell lines were maintained in RPMI1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 50 U/mL penicillin and 50 μg/mL streptomycin. All cell lines were incubated at 37 °C with 5% CO2.

In vitro Cell Viability Study
Cells were seeded in 96-well plates for 24 h at 37 °C with 5% CO2. SKBR3 cells were seeded at 15,000 cells/well, BT474 cells were seeded at 20,000 cells/well, MCF7 cells were seeded at 7,500 cells/well and MDA-MB-468 cells were seeded at 10,000 cells/well. Serial dilutions of trastuzumab, C-ADC and NC-ADC were added to the cells in complete growth medium and incubated at 37 °C with 5% CO2 for 96 h. Cell viability was measured using CellTiter-Glo viability assay (Promega) according to the manufacturer's instructions. Cell viability was plotted as a percentage of untreated cells. Each measurement was taken in triplicate and three independent repeats were performed.

In vivo Efficacy Study
All animal experiments were carried out at the Biological Resource Unit at Cancer Research UK (CRUK) Cambridge Institute. The experiments were performed in accordance with the UK Animals (Scientific Procedures) Act 1986, with approval from the CRUK Cambridge Institute Animal Ethical Review and Welfare Body. Female NSG mice (6-8 weeks old) were purchased from Charles River. The animals were verified as pathogen free and in excellent health.
Subcutaneous BT474 xenografts were established by implantation of 10 7 cells in PBS/Matrigel (1:1, 200 µL). From the day of implantation, the drinking water was supplemented with bestradiol (Sigma-Aldrich) (5 µg/mL, 0.1% ethanol). When tumour volumes reached 200 mm 3 (4-8 weeks after implantation), mice were randomised and enrolled to the study (n = 3 per study arm). Mice received two, weekly tail vein intravenous bolus injections of trastuzumab, NC-ADC, C-ADC or vehicle (PBS). Bodyweight and tumour volumes were measured 2-3 times per week for 60 days after enrolment. Tumour volume was measured using a Vernier caliper and was calculated as follows, where l is equal to the length (mm) of the longest tumour side and w is equal to the width (mm) of the shortest tumour side.  Table S1) was performed with a multiplicity adjustment allowing to take the dependence between the contrasts of interest into account when setting the family-wise error rate at a 5% level (R function multcomp::glht). To compare the average tumour volume at day 60 of the different groups with respect to the reference one, 100 ul PBS, we first fitted a linear model (R function stats::lm) with the response on the cube root scale and 'group' as predictor, and then analysed the contrasts of interest (linear combinations of regression parameters, ESI Table S2.) using a Dunnett-type multiplicity correction allowing to take the dependence between the different comparisons of interest into account when setting the family-wise error rate at a 5% level (R function multcomp::glht). Sensitivity analyses considering heteroscedastic modelling led to the same conclusions. Trastuzumab @ 10mg/kg vs PBS -5.112403161 0.0007 *** NC-ADC @ 1 mg/kg vs PBS 0.727636105 0.9622 NC-ADC @ 10 mg/kg vs PBS -7.343271533 <0.0001 *** NC-ADC @ 20 mg/kg vs PBS -7.342875813 <0.0001 *** C-ADC @ 1 mg/kg vs PBS -0.462630976 0.9967 C-ADC @ 10 mg/kg vs PBS -6.570414028 <0.0001 *** C-ADC @ 20 mg/kg vs PBS -7.340337957 <0.0001 ***

In vivo Selectivity Study
Female NSG mice (6-8 weeks old) were purchased from Charles River. The animals were verified as pathogen free and in excellent health. Subcutaneous BT474 xenografts were established by implantation of 10 7 cells in PBS/Matrigel (1:1, 200 µL). Subcutaneous MCF7 xenografts were established by implantation of 10 6 cells in PBS/Matrigel (1:1, 200 µL). From the day of implantation, the drinking water was supplemented with b-estradiol (Sigma-Aldrich) (5 µg/mL, 0.1% ethanol). When tumour volumes reached 200 mm 3 , mice were randomised and enrolled to the study (n = 3 per study arm). Mice received a tail vein intravenous bolus injection of trastuzumab, NC-ADC, C-ADC or vehicle (PBS). 72 hours after treatment, mice were euthanised. Tumours and organs were excised and fixed with 10% neutral buffered formalin (NBF). Upon fixation, tissue samples were embedded in paraffin and stored for histology. H&E, TUNEL staining and Ki67 was carried out as standard.

Pharmacokinetics Study
Female NSG mice (6-8 weeks old) were purchased from Charles River. The animals were verified as pathogen free and in excellent health. Subcutaneous BT474 xenografts were established by implantation of 10 6 cells in PBS/Matrigel (1:1, 200 µL). 7 days after implantation, mice were randomised and enrolled to the study (n = 3 per study arm). Mice received a tail vein intravenous bolus injection of NC-ADC or C-ADC. Blood samples were collected 2, 6, 24, 72, 120 and 168 hours after treatment. K2EDTA (0.5 M in PBS) was added to each sample to a final concentration of 1.8 mg/mL of blood. Samples were stored on ice before centrifugation at 1600´g at 4 °C for 10 min. The plasma fraction was removed and stored at -80 °C until analysis.  The peak area ratios from the integrated analyte and internal standard chromatograms were used to back calculate the concentrations of Trastuzumab against a quadratic 1/x2 weighted calibration curve using MultiQuant™ v3.0.3 software (AB Sciex LLC, Framingham, USA).
Sample analysis was conducted in a batch, consisting of calibration standards, quality controls (QCs), blank control matrix samples, internal standard only (zero sample) and study samples.
Precision and accuracy of the QCs were within the acceptance criteria of ±20%. Time versus concentration data was imported into PCModfit V6.0 software using a Microsoft Pharmacokinetic parameters were estimated using non-compartmental analysis.